Project Summary Enzymes that install posttranslational modifications (PTMs) on bacterial peptides and proteins are integral in cellular functions such as the formation of bioactive peptide natural products and the activation of enzymes important for bacterial adaptation to oxygen-limited environments. Understanding the process of PTM formation can inform on the engineering of novel peptide therapeutics and on the methods of bacterial colonization of host environments in infection. S-adenosyl-L-methionine (AdoMet) radical enzymes produce numerous PTMs that change the functionality of the targeted residue(s). AdoMet radical enzymes perform oxygen-sensitive, site-selective radical chemistry on macromolecular substrates, yet a structural understanding of how they accomplish this impressive chemistry has lagged behind in the analysis of the AdoMet radical enzyme superfamily, with no complete AdoMet radical enzyme-protein complex fully visualized. The aims of this proposal include structural characterization of two AdoMet radical enzymes that modify the C? of specific amino acids within their large substrates: 1) an AdoMet radical epimerase with a peptide substrate and 2) pyruvate formate lyase activase (PFL-AE) in complex with its partner PFL. The epimerase irreversibly converts L-amino acids to D-amino acids within a ribosomal peptide, thus altering the final conformation and influencing its bioactivity. Determining how one epimerase positions substrate to perform multiple turnovers at specified residues will require structural insight. X-ray crystallography will be used to examine interactions of AdoMet radical epimerases with peptide substrates. PFL-AE forms the catalytically essential glycyl radical on PFL to make formate and acetyl-CoA from pyruvate and CoA. How PFL-AE contacts PFL and how the glycyl radical transitions from the PFL-AE active site to the buried PFL active site remain to be elucidated. X-ray crystallography and electron microscopy will be used to determine structures of PFL-AE in complex with PFL. Structural analysis of both systems will provide much needed insight into interactions required for construction of a protein complex that performs site-selective oxygen-sensitive radical-generating chemistry.